Drying of yeast to inactivate zymase and preserve lactase



United States Patent Edwin G. Stimpson, Sayville,.N. Y., assignor toNational Dairy Research Lalio corporation of nenwaie.

No Drauiosi Al Rli hfiqll No ember 19.52. Serial No. 322,580

2 claims. Cl. 195-65) 'Th s inv ntion rela es t he produ ym a d, moreP.e "1 lo l a lactase-active' zyiiia ma fl r n goo S abil ty, o by thisprocess. v The utilization of milk products in concentrated form hasbeen limited heretofore by the low solubility of lactose in water. Forexample, skim milk cannot be Stored if it. is concentrated beyond atotal solids content of for at higher concentrations large lactosecrystals q y e eposited. Similarly, there is an upper limit of solidsconcentration for both whole mill; and whey beyo w i h lactose c ystalization becomes" it Also, in the manufacture of ice earn tsolids-not-fat must be withinthe r o t h e pro o on ac ose and 'producea so-called sandy 1 Crystallization of lactose is'a set it upsets theuniformity of the pro ct and product difiicu'lt to' handle and use. Thep; le'm 7 further complicated by the ditiicul't'y experienced inv if Prom. e a se dissolving lactose once'it has crystallized For this reason,it has been proposed that the lactose present in milk be" hydrolyzed tosimple sugars" 'Qe Soluhle in w e h lactos h'ro fhse or lacto enzyme. Insuch. aprocess the lactas'e' enzymeprepara tion is added to' the milk,which is then incubated 'at an elevated temperature until the desiredamount of hydrolysis has taken place? A c r y it s a object of thinvention to provide n mp o d m od of preparing a lactase enzymepreparation which can be empl yed to hydrolyze the lact se present inmill; and produce a nearly or substantially loc ree milk product o g oflavor.

It s f h an jec of the invention to prepare a lactase-activezymase-inactive yeast product of bland flavor and good stability.

These j t ar i o t ishe l in c ordan e with the nve n y P op gatingyeast of a a asep oducin ain under g ow h-fl o ing conditi ns in anutrient medium i i h lac os s the'chi f s ur e f arbohydrate and thentreating the yeast to inactivate the zyrnase.

A o h y ast st ai w ich may be p opaga ed. by the process of theinvention hr? Saccltargmycgs fragilis, Toru'lopsis spherica,Zygosaccharomyces lactis, and strains of Torula utilis and, Candidapseudotropicalis adapted to the utilization of lactose for. grow h andfiermentation.

The ingredients and proportions thereof essential to any nutrient mediumemployed for the propagation of yea t nd p rt ul r y yeasts of the abovestrains, are well known to those skilled in the art and details thereofneed not be given here. It is important, however, that n en dia employedin accordance with the present invention contain lactose as the chiefsource of carbohydrate in an amount within the range from 0.5"t0 5 Thetotal solids content of the nutrient medium should be between about 2and about 8%, preferably 3%, and its pH should be approximately 45, buta satisfactory enzyme preparation can be obtained employing a nutrientmedium whose'pH lies within the range of 3.5 'to 7.5."

The nutrient medium may be prepared'synthetic'ally by dissolving inwater 0.5 to 5% lactose, 0.4% ofs'ome source of inorganic nitrogen, suchas urea, ammonia, or diammonium phosphate, 1% peptone, 0.5% yeastextract, and other yeast nutrient materials. Usually it'is'""inpreconvenient, however, and lessexpensive', to employ" as'a rritories,Inc., Galrdal, N. 35., s

2,693,440 Patented Nov. 2, 1954 base for the. medium a natural productwhich contains all or a la e. Pr port n o he ma e a eq for Yea row h; anto s pply nu r e s n whi e e i l they e cient. Well nown mat r in p iUP- ply which contain all yeast nutrient requirements except inorganicnitrogen are whey derived from cheese or case n manufacture, and themother liquor wash Water obtained as. aste produ in the pro uc ion of at from e or milk products.

In the table there is given the typical composition of some whey andwhey derivatives useful as a base for the v stn t ent m d Table MotherQheddar Cream Casein Liquor Cheese Cheese Whey Wash Whey Whey waterPercent Percent Percent Percent Tot 1S olids 3.0 3.0 3.0 3.0 Lac ateIon;; 0.11 0.26 0. 10 0. 24

It has been determined that it is important that the nutrient medium bepasteuri'zed'before yeast is added thereto. Pasteurization may bebrought about by heating" the medihfn' at 14Q'to'1'60" F. for 30 minutesor lon e l or [for /:hour, or at to F. or hig 'er'fo'r l0 to'30 seconds.Consoq l l fly, when whey is employed as the yeast nutrient medium inthe process of the invention, while the whey isk'ept at an elevatedpasteurization temperathr'e, it is 'f ort'ified with such additionalyeast nutrient materials assay be required. It is desirable to add thesematerials at a pasteurizationtemperature in order roy bacteria'containedtherein. Some source of itr'o' en should'be added, say 0.2%urea, 0.14% 1a, or. 4% diammonium phosphate". 'Also, if Q red, 0.1%corns ep'may be added. The solids con the whey is then" adjusted towithin the rang "Dependent upon the ifiitial solids coriccomplishe'deither in the'course of fortifyi'ng with additional 'solid'nutrientrnaterial or by dilution withwatfir Or a solution of the addednutrient mateflaliw. "Berore addition of yeast, the nutrient medium isbrought to a'p'r'opagatiori t mperature within the range ofSQ' to "100E."Ah activel'y'growing starter culture of the yeast" in the nutritivemedium then is added in an amountof 10% on "thebasis of the'main batchof medium." This inoculation should give a main fermenta- P Be h laihihga ce oun of 10 to million Yea t ce s p r ml. D rin ie mentation henutrient edi m he d' at. a. emperature within this range; a temperatureof 8d? F. is the. optimum.

I n as ation. i m o ed 3 r r m re m y bereqhi e for erme tion There or io de to' shorten the fermentation time it is desirable to aerate the nttrient medium. Aeration at a rate of 0.009 volume ofalir'per vfun'ie'of nutrient medium per minute will re e'the r m. h'tim o m .8 tohours, while a higheraer n' rate, of the order of 0.5 volume of air pervolume of nutrient medium per minute, will tu her'r dn' etheiermeniationtime tO'frQm 10 2 ho s; In general, the'hig ier thefrat'e oraeration, vvithn the range d scr on rates spe ified, the h her he Yi l oyeast. Wh e an aeration atewi h n the r n of 0.009 to 0.5 volume ofairper volume of nutrient In dium per minute is preferred; it will beunderstood h hi a re ohr s may emp oyed t me Special mg l te'n from 2 to8%; thin; h may h a If, in addition to aeration, the nutrient medium isagitated vigorously, it is possible to further reduce the fermentationtime to from 2 to 8 hours.

With a combination of aeration and agitation, t is possible to establisha continuous fermentation process in which the yield of yeast in a unittime 18' at least times that of a batch process under similargrowth-favoring conditions. In this process, nutrient medium is fed inat a constant rate to replenish exhausted nutrients, while fermentednutrient medium is withdrawn at a similar constant rate for theharvesting of yeast therefrom. Such a continuous process is particularlydesirable from a commercial point of view.

The nutrient medium is the same as is used in a batch process, andpreferably is derived from whey. In practice, the nutrient medium isinoculated with wet yeast cake in an amount equal toone to two billionyeast cells per cc. (0.7 to 1.4%, by weight of the medium based on thedry weight of theyeast added), and these cells are encouraged to grow ata maximum rate under optimum growth-favoring conditions of aeration (0.1to 1 volume of air per volume of nutrient medium per minute), agitation(sutficient to give a dispersion of small air bubbles throughout theliquid), and temperature (80 to 100 F.) using proper rates of feeding inof nutrient medium and withdrawing of fermented liquors.

A yeast concentration of one to two billion cells per cc. representsapproximately the maximum concentral temperature of about 170 F. areoptimum conditions for tion which can be maintained withoutovercrowding.

Therefore it is desirable in a continuous process to maintain theconcentration Within this range by removing fermentatioirliquorcontinuously at the same rate at which fresh nutrient medium is added.

fYeast may be harvested from this withdrawn liquor continuously or fromtime to time, as desired. Spent liquors may be fortified with nutrients,and reused.

At 86 F., with proper agitation and aeration at a rate of 0.2 volume ofair per volume of medium per minute, using a whey-base nutrient mediumof 3% total solids, fortified with 0.4% diammonium phosphate, an initialinoculation of one billion actively growing S. fragilis yeast cells percc. of medium, a continuous fermentation will double itself to form 2billion yeast cells per cc. every 4 hours, whereas a batch fermentationunder the same conditions would produce only 0.15% of yeast cells or amaximum of 200 million yeast cells per ml. in the same time and not over800 million yeast cells per ml. in 18 hours.

In either the batch or continuous processes, if a product of superiortaste properties is desired, it is advisable to wash the yeast in waterafter it has been harvested. For most uses, however, as in thehydrolysis of lactose for the preparation of an animal feed or as anutrient in antibiotic media, washing is not essential.

The harvested yeast contains a highproportion of lactase and zymase. Thelactase enzyme preparation is intended to be used in the hydrolysis oflactose to glucose and galactose without converting glucose andgalactose to carbon dioxide and alcohol, and it is essential'that'thelactase be uncontaminated with those yeast enzyme systems which convertglucose and galactose to carbon dioxide and alcohol. These enzymesystems are collectively called zymase by the art, and it will beunderstood that yeast lactase preparations of the invention must bezymase-inactive if conversion of glucose and galactose to carbon dioxideand alcohol is to be prevented. If the zymase content in the yeast isinactive, it is not necessary to separate the lactase from the yeastbefore adding the preparation to the milk product.

The zymase may be destroyed without destroying the lactase by drying theyeast under carefully controlled conditions. v

Spray-drying under carefully controlled conditions produces a product ofhigh potency. The spray-dried product likewise has a good flavor and agood color, and does not impart an adverse flavor to a milk product.

It is recommended when spray-drying is used that the yeast be dispersedin water to form a cream or slurry of from 10 to 18% yeast solids. Thisslurry is fed into a spray drier whose inlet air stream is from 200 to310 F. and whose outlet air stream is from 130 to 205 F. At temperaturesabove this range there occurs a 40 to 65% loss of enzyme activity, Whileat temperatures below this range it is not possible to obtain a dryenough product; An inlet air temperature of 250 F. and an outlet airTest:

most spray driers. The dry yeast powder should be cooled to roomtemperature as quickly as possible after leaving the spray drier andshould then be stored at 40 F.

A satisfactory product can be prepared by drying the yeast in a traydrier at a temperature not in excess of 150 F. If the drying is carriedout at atmospheric pressure, the drying time should not be more than 4hours, but if a vacuum is employed a drying time of as long as 6 hoursis tolerable. Drying times up to 8 hours have been used under conditionsof high relative humidity or where the temperature employed is about 115F. The tray-dried product is darker in color than the spray-driedproduct.

Drying on a roll drier completely inactivates the lactase enzyme.

The lactase preparation in accordance with the invention is white, greyor tan in color, depending upon the drying process employed, andpreferably will be in powder form or in the form of small friable lumps.The preparation disperses readily in three times its volume of water. Ithas the following analysis:

Percent Moisture 4 to 10 Protein (N x 6.38) 40 to Ash 7 to 10 Fiber 3 to10 The lactase activity (and zymase inactivity) of the lactasepreparations of the invention is determined by the following test,hereinafter called the Lactase Activity The preparation, when mixed with30% total solids, pasteurized, nonfat milk concentrate in a proportionof 1 part of preparation to each 40 parts of lactose, shall give 85%conversion of the lactose therein to simple sugars in 4 hours time at123 F. A technical grade of the lactase preparation such as might beused in the preparation of animal-or poultry feeds would be consideredacceptable within the requirements of the Lactase Activity Test if itgives conversion of the lactose to simple sugars within such time at thetemperatures stated.

Lactase preparations in accordance with the invention can be prepared asset forth in the following examples:

EXAMPLE 1 Whey derived from the production of casein or Cheddar cheeseor cream cheese is adjusted to a pH of 4.5, either by addition of lacticacid or by treatment with lactic acidproducing bacteria. The whey isthen heated at 185 F. for 30 minutes in order to coagulate the protein,and the coagulated protein is separated by decantation or filtration.

While the clarified whey is still at 185 F., 0.4% diammonium phosphateis added, and the pH of the whey is again brought to 4.5. The solidscontent is now adjusted to 2 to 8%. After cooling the whey to 85 F.,from 0.2 to 0.4% of the whey of S. fragilis wet yeast cells are added,and the mass is allowed to ferment for about 20 hours at approximately86 F., with aeration with from 0.009 to 0.5 volume of air per volume ofmedium per minute.

The yeast cells are separated from the fermentation liquor and washedwith warm water.

The yeast is then dispersed in water to form a yeast cream of from 10 to18% solids content. The yeast cream is fed into a spray drier whoseinlet air stream is at a temperature of about 250 F. and whose outletair stream is at approximately 170 F. The dry yeast powder is cooledtoiroom temperature as quickly as possible after leaving the spray drierand is stored at 40 F. until use.

The lactase enzyme preparation obtainable by the above process has goodstability, and passes the Lactase Activity Test. It has a good lightcolor and a bland flavor, and does not impart an undesirable flavor tomilk products in whichit is incorporated.

The following example illustrates utilization of this product tohydrolyze the lactose contained in a milk product:

EXAMPLE 2 and the resulting, skim milk pasteurized at 160 F. for

30 minutes, then condensed in vacuo at a low temperature to a 30% solidscontent. The temperature of the condensed skim milk is adjusted to 123F. Yeast lactase, prepared as set forth in Example 1, is dispersed infour times its weight of water to form a slurry, and this slurry isadded to the skim milk in a ratio of one part by Weight yeast lactase toeach fifty parts by Weight of lactose in the skim milk. During additionof the lactase slurr the skim milk is agitated vigorously. The mixtureis then held at 123 F. for four hours, at the end of which time 80 to90% of the lactose has been converted to glucose and galactose. In orderto inactivate the lactase enzyme, the mixture is heated at 160 F. for 30minutes. The product is cooled and may be frozen and stored at 0 F., orit may be dried by any convenient method, such as in a roll or spraydrier.

The dried product may be used to prepare an ice cream or a processcheese.

EXAMPLE 3 Whey derived from the production of casein or Cheddar cheeseor cream cheese is adjusted to a pH of 4.5 by addition of lactic acid.The Whey is then heated at 185 F. for 30 minutes, after 'which 0.4%diammonium phosphate is added, also at 185 F. and the coagulated proteinis separated by filtration. The solids content of the whey is adjustedto 2 to 8%.

To the deproteinated whey is added 0.2% by weight of the whey of Torulopsis spherica yeast cells and the mixture is held for 30 hours at86 F. without aeration. The yeast cells are separated from the Whey,washed in warm water and then placed in an atmospheric tray drier Whosetemperature is not over 150 F., but high enough and with sufficient aircirculation to dry the yeast completely in less than 4 hours. The dryyeast powder is cooled to air temperature as quickly as possible afterleaving the drier and should be stored at 40 F.

This product passes the Lactase Activity Test, has a good color and canbe used as an additive to milk products in order to hydrolyze thelactose thereof to glucose and galactose.

In contrast, when the yeast is dried above 150 F., or for over 4 hours,the product may not pass the Lactase Activity Test, it has a dark colorand may be expected to display substantially less lactase potency thanthe freezedried product. Moreover, this product imparts a veryobjectionable flavor to the milk product. If the yeast is dried on aroll drier, the lactase enzyme appears to be completely inactivated, andthe product does not pass the Lactase Activity Test.

EXAMPLE 4 Whey derived from the production of Cheddar or cream cheese isadjusted to 8% solids content and inoculated with lactic acid-producingbacteria, after which it is incubated until the pH has reached 4.5. Itis then heated at 185 F. for 30 minutes in order to coagulate theprotein. After separation of the protein 0.2% urea is added, and thenthe whey is pasteurized by heating at 145 F. for 45 minutes. The pH isthen adjusted to approximately 7.0 and 0.1% by weight of the whey of S.fragilis yeast cells are added.

The mixture is allowed to ferment for 10 hours at a temperature ofapproximately 86 F. with aeration at a rate of 0.5 volume of air pervolume of whey per minute.

The yeast is separated from the whey, washed with warm water, anddispersed in water to form a slurry of 10% solids content. The slurry isdried in a spray drier with an inlet air stream at a temperature of 250F. and an outlet air stream at a temperature of 170 F. The dried powderis cooled to room temperature as quickly as possible and then stored at50 F. The dry yeast passes the Lactase Activity Test, has a good colorand a bland flavor and may be stored at 40 F. for long periods Withoutfurther loss of activity.

In contrast, yeast spray-dried at an air inlet temperature of over 310F. and an air outlet temperature of 205 F. suffers a 40% loss of enzymeactivity and yeast spray-dried at an air inlet temperature of 340 F. andat an air outlet temperature of 220 F. suffers a 65% loss of enzymeactivity. Neither of these products passes the Lactase Activity Test. Onthe other hand, if the inlet air temperature is 200 F. and the outletair temperature is 130 F., a dry enough product is not obtained.

EXAMPLE 5 The continuous fermentor employed in this example is designedto allow for a constant input of nutrient medium and simultaneous drawoif of fermented liquors. The fermentor may be equipped for ampleaeration and agitation with a centrifugal pump impeller revolving belowa central draft tube extending upwardly from the bottom to two-thirdsthe height of the fermentor. Air may be injected at the bottom of thevat and the in peller distributes the air in small bubbles throughoutthe medium contained in the vat. Where there is a possibility of the airbeing contaminated with microorganisms, it is advisable to filter orsterilize the air be fore injecting it into the vat. When using such afermentor, any foam produced is sucked down the central draft tube andredispersed, thus preventing the tank from foaming over.

Fermented liquor passes continuously through an overflow pipe into asurge tank where it is stored ready for further processing. From time totime the mate rial in the surge tank is centrifuged to harvest activelactase-containing yeast therefrom. H j

For a daily run, the pH of a 100 lb. batch of Cheddar cheese Whey isadjusted by addition of lime and 0.1% calcuim chloride to 6.8. The wheyis heated by steam injection to a temperature from 185 to 200 F. andallowed to stand at this temperature from one to two hours.Heat-coagulable protein will precipitate and separate to form an upperlayer of clear deproteinated whey, approximating 75 to 80% of the mass.This upper layer is decanted and pumped to the fermentor.

Any additional nutrient materials are added While the decanteddeproteinated whey is still at about 200 F., in order to take advantageof the bactericidal characteristics of this high temperature. To theapproximately 96 lbs. of clarified whey, taking into account dilutionfrom steam injection, obtained from the original bath of 100 lbs., thereis added 0.64 lb. of diammonium phosphate to produce a nutrient mediumcontaining 0.4% diammonium phosphate by weight. This mixture is dilutedwith 64 lbs. of cold water or of centrifugate of a previous run toproduce the nutrient feed medium. The medium is now at a temperature ofapproximately F., and this temperature is maintained during storage toavoid bacterial spoilage.

To start the fermentor, approximately 1.6 gallons of the nutrient feedmedium is brought to 86 F. and placed in the fermentation vat.Temperature controls are adjusted to maintain this temperature, aerationis begun at a rate of 0.13 ft. /min., and the impeller is started up. Aslurry of approximately 0.18 lb. of S. fragilis yeast cells in water isadded.

The progress of the fermentation is followed by analysis of samples ofthe nutrient medium for lactose content. The fermentor is operated untilthe lactose content has been reduced to 0.1 or 0.2%; this requires fromtwo to six hours. At this time the yeast cell count will be over 1billion cells per ml. From now on the fermentor may be operatedcontinuously.

Nutrient feed medium at 86 F. is led into the fermentor continuouslyfrom the feed tank at a rate of 0.0133 gal/min. and aeration iscontinued at a rate of 0.13 ft. /min. After about two hours thefermentor will be full and fermentation liquor will run out the overflowpipe into the surge tank. From time to time the yeast cells areharvested by centrifuging the material collected in the surge tank, andthe centrifugate therefrom may be stored and later fortified withnutrients and fed back into the nutrient feed medium. The fermentat1onmay be continued for days at a time without interruption, provided thatthe pH is maintained at from 4.5dto 5.5 by addition of either ammoniawater or lactic ac1 Under these conditions 19.2 gallons of dilute wheycan be processed daily, producing 4.8 lbs. of wet yeast cake, which isreduceable by spray-drying, as set forth in Example 1, to 1.2 lbs. perday of a dried active lactase preparation which passes the LactaseActivity Test.

As illustrated in Example 2, the lactase-active zymaseinactive enzymepreparation of the invention may be utilized to hydrolyze the lactosecontained in a milk product in order to produce a substantiallylactose-free product whose total carbohydrate content is undiminishedcompared to the natural product, and which has sufiered no deteriorationin flavor or appearance by the treatment. Thus the preparation may beused for preparation of ice cream concentrates derived from skim milksolids which do not contain lactose and which therefore do not developsandiness. It may also be used to prepare animal feeds which contain alarge amount of milk solids but are free from the cathartic efiect oflactose.

Various modifications and changes may be made in the conditions underwhich the process of the invention is carried out, as will be apparentto those skilled in the art, and it will be understood that theinvention is not to be limited, except as set forth in the followingclaims.

All parts and percentages in the specification and claims are by weight,unless otherwise indicated. Percentages of components of the nutrientmedium or of the whey are by weight of the nutrient medium, or of thewhey, respectively.

This application is a continuation-in-part of copending application,Serial No. 224,252, filed May 2, 1951.

I claim:

1. A process of producing a lactase-active zymaseinactive yeastpreparation which comprises spray drying an aqueous dispersion oflactase-active zymaseactive yeast of a lactase-producing strain at atemperature References Cited in the file of this patent UNITED STATESPATENTS Number Name Date 1,391,562 Miller Sept. 20, 1921 FOREIGN PATENTSNumber Country Date 474,822 Great Britain of 1936 OTHER REFERENCESOppenheimer et al., Ferments and Their Actions, Chas. Griflin andCompany Ltd., 1901, pages 204-205.

Dienert, Comptes Rendus 129, 1899, pages 63-64.

1. A PROCESS OF PRODUCING A LACTASE-ACTIVE ZYMASEINACTIVE YEASTPREPARATION WHICH COMPRISES SPRAY DRYING AN AQUEOUS DISPERSION OFLACTASE-ACTIVE ZYMASE-ACTIVE YEAST OF A LACTASE-PRODUCING STRAIN AT ATEMPERATURE WITHIN THE RANGE FROM 130 TO 310* F. TO INACTIVATE THEZYMASE AND RECOVER A LACTASE-ACTIVE ZYMASE-INACTIVE PRODUCT.